Be patient; some of these movies will take a while
to load. You may want to do this using two browser windows, one for
looking at movies and one for reading text.

Saccoglossus 040923 explants

Each set described left to right, top to
bottom

AS = animal cap sandwich

VS = vegetal plate sandwich

SR = Sagittal ring = a torus including both animal
and vegetal

SH = Sagittal Hemisphere = half an embryo cut
sagittally

VP = vegetal plate; the part that
invaginates

MZ = marginal zone (arbitrarily define as the
region next to the VP)

AC = animal 1/2 or so

BP = Blastopore

Sacco
sand group 2: Upper row: AS, VS, VS; lower
row: AS, VS, AC. The AS's don't do much; somewhat remarkably, they
neither swim away nor rotate; ciliary beat in the opposite direction
from the two halves? The VS's don't do much either, though the one in
the middle of the bottom row shows an odd behavior; this VS is not
well matched with its other half, underneath (esp. at lower right);
notably, in the region toward the center from this a hole
spontaneously opens up and many cilia or perhaps filopodia appear on
the inner side of the hole. This hole pretty clearly forms at the
interface between the vegetal plate and the marginal zone, where one
imagines the cleft that forms on the basal surface would be. The
other two VS's grow some cilia, and wind up with lots of loose cells
on the surface (which may be unhappy or dying cells). The animal cap
closes it's open, basal side a bit, spins around for a while, then
seems to stop and flatten out (maybe it's dying?). Not a real
exciting experiment.

Sacco
set 1: This is actually Ray's handiwork.
Upper row: SR cut animally, SR cut vegetally, and an SH; Lower row:
two uncut SR's, and another SH. Everything starts vegetal down. First
frame is 4.5x, then all 5x. At frame 8, I've pried the two cut SR's
apart; they show that the buckling of the VP is pretty autonomous.
Not entirely clear why the vegetally cut SR opens as it does; perhaps
the AC region generates tension apically? Note that the animally cut
SR makes a serious attempt to actually close it BP on all sides. Not
sure why the first SR shows fairly good BP closure, while the second
does not. The two SHs manage to close their blastopores fairly
effectively; although the details aren't particularly clear, it looks
like they do so fairly evenly, and with about the some time course as
an intact embryo, suggesting that the contraction of the marginal
zone will opperate to close the BP almost as effectively as in an
intact embryo, i.e. the continuity of a ring of contraction within
the marginal zone is not necessary. On the other hand, the two SRs do
a lousy job of closing their BP, so maybe half an embryo worth of MZ
is enough, where as two small (1/8th circumference?), separated bits
of MZ are not. The SHs also appear to undergo some animal-vegetal
elongation.

Sacco
set 4: My attempt to play with SR's;
obvious what's what; all start VP down. Not real interesting. Don't
see the opening of the vegetal side in the vegetally cut SRs, as in
set 1; the ends of the vegetal plate seem to want to come back
together when pried apart. Maybe I need to make these earlier during
BP formation, as in set 1. Especially in the third vegetall cut SR,
the bending vegetal plate actually seem to bend the adjacent MZ
outward. Animally cut SRs consistently show BP closure behavior,
showing that an intact AC is not required.

The first explant is notable because the MZ
appears to contract, or converge toward the top, in the
circumferential direction; this behavior begins about frame 50 is
strongest as the blastopore is contracting in the whole embryo. The
VP begins moderately dimpled, but then flattens out and never
invaginates. This and other experiments suggest that the entire MZ is
programmed to converge circumferentially as the VP invaginates.

The second explant balls up and swims off.

The third explant invaginates nicely, and tries to
close the BP, but is much delayed w.r.t. the whole embryo. With the
first explant, this suggests that a circumferentially intact marginal
zone is required for invagination.

The whole embryo shows the normal progression of
blastopore closure, as a controls for the behavior of explants in
position 1, 3 and 6.

The final explant tries to invaginates, but
notably, does not do nearly as well as the VP with more MZ; it seems
as though its trying to close its BP (see frames 80-150). There are
many highly active ("loose"?) cells; perhaps VP cells have a more
mesenchymal character than MZ and AC cells, or perhaps the VP cells
are more unhappy about being exposed than MZ and AC cells, or maybe
its just a generally unhappy explant. But see also the cells at the
lower edge of the first explant, which also show a lot of
activity.

Conclusions:

An intact AC is not required for VP invagination
or (at least some) BP closure.

The marginal zone tends to converge
circumferentially, and appears to be required for VP
invagination

Questions:

1) what is the A-V extent of the contractile
MZ?

_________________________________________________________________

Saccoglossus movies 040924

all embryos nile blue labeled; all embryos pinned
in place with coverslip bits. "sagittally" = cut along the AV
(Animal-Vegetal) axis, rather than transversely, across the AV
axis.

epi
01 VP basal** a vegetal plate, looking at
the basal surface; note in particular the peri-plate tissue
converging toward the lower left (between about 6 and 8 O'clock);
also, between frames 10 and 20, a depression forms in this region
between this tissue and the plate proper, but largely dissappears by
100. Lots of protrusvie activity/blebs at free edges.

epi
02 VP apical a vegetal plate from the
animal side (or possibly it's an animal half?); a curious wave
propagates inward from the lower right, from about frame 35 to 90;
also more subtly from the upper margin of the explant, at the same
time. Lots of cilia at the margins, especially upper left. Boring
otherwise.

epi
03 VP basal*** The piece de resistance: A
vegetal plate from the basal side; a very clear division/cleft forms
around the vegetal plate, except on the right (presumably, I cut more
vegetally there); clearly visible by frame 50 and persists
throughout; the tissue outside the cleft converges to the left, and
gets "thicker" in the Animal vegetal direction, presumably reflecting
isotropic (circumferentially equal) convergence and extension in the
non-involuting tissue. Doesn't really seem to drag a lot of the
vegetal plate cells with it as it converges, though it does leave
behind a free edge that seems to be more protrusive than the
converging marginal zone.

epi
04 BP vegetal view of BP closure; embryo
was held under glass; BP closure seems inhibited (perhaps pressed too
hard? Suggests that Animal-vegetal extension is required as part of
the process); perhaps some isotropic convergence (could try measuring
it, but .), but no obvious rearrangement.

epi 05 BP as 04, but even less exciting

epi
06 BP as 04, but some weirdness going on
just inside the blastopore

epi 07 BP ditto 05

epi
08 ring a "sagittal" ring (AP is at 11:00,
VP at 5); looks too squished under the CS; does sort of flatten up
and fold in on itself

epi
10 hemi A half embryo, cut "sagittally";
not pretty, probably under too much pressure from CS; vegetal plate
sort of everts; does try to close up the opening, dunno what that
means. Some animal-vegetal extension.

epi
11 hemi* as for 10; I don't really
understand what the vegetal plate is doing, but it does it here too.
There is also some fairly strong animal-vegetal extension evident as
well.

_________________________________________________________________

040925 Saccoglossus movies

These explants were made as the embryos became
clearly dimpled. They are either "animal caps", which don't include
any vegetal plate, and little of the region near it; "vegetal
plates", usually partial plates with some attached "marginal zone"
material; or "planula 1/2's", cut sagittally and also pinned under
coverslips (but still wander off).

epi
16*: Smaller chunk of VP, with cleft, in
upper right, attached to larger bit of MZ & AC; not dramatic, but
a very clear waist forms at the MZ region! Not sure which side is
visible; cleft heals rapidly & early.